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Contents lists available at ScienceDirect

Plant Science

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ontrolling stomatal aperture in semi-arid regions—The dilemma ofaving water or being cool?

.M. Chaves a,∗, J.M. Costa a,b, O. Zarrouk a, C. Pinheiro a,c, C.M. Lopes b, J.S. Pereira b

Plant Molecular Physiology Laboratory, ITQBNOVA, Universidade Nova de Lisboa, Oeiras, PortugalLEAF, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, Lisboa, PortugalFaculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica 2829-516, Portugal

r t i c l e i n f o

rticle history:eceived 16 February 2016eceived in revised form 14 June 2016ccepted 22 June 2016vailable online xxx

eywords:tomata

UEeaf temperatureight-time transpiration

a b s t r a c t

Stomatal regulation of leaf gas exchange with the atmosphere is a key process in plant adaptation to theenvironment, particularly in semi-arid regions with high atmospheric evaporative demand. Developmentof stomata, integrating internal signaling and environmental cues sets the limit for maximum diffusivecapacity of stomata, through size and density and is under a complex genetic control, thus providingmultiple levels of regulation. Operational stomatal conductance to water vapor and CO2 results fromfeed-back and/or feed-forward mechanisms and is the end-result of a plethora of signals originated inleaves and/or in roots at each moment. CO2 assimilation versus water vapor loss, proposed to be thesubject of optimal regulation, is species dependent and defines the water use efficiency (WUE). WUE hasbeen a topic of intense research involving areas from genetics to physiology. In crop plants, especially insemi-arid regions, the question that arises is how the compromise of reducing transpiration to save water

rop managementemi-arid regions, Heat wave

will impact on plant performance through leaf temperature. Indeed, plant transpiration by providingevaporative cooling, is a major component of the leaf energy balance. In this paper we discuss the dilemmaof ‘saving water or being cool’ bringing about recent findings from molecular genetics, to developmentand physiology of stomata. The question of ‘how relevant is screening for high/low WUE in crops forsemi-arid regions, where drought and heat co-occur’ is discussed.

© 2016 Published by Elsevier Ireland Ltd.

. Introduction

Under semi-arid climates (receiving precipitation below poten-ial evapotranspiration) plants are often subjected to periods ofater deficits, with high impact on plant functioning and produc-

ion. The effects depend on stress duration, intensity and rate ofrogression, as well as on genotype, developmental stage of plants

Please cite this article in press as: M.M. Chaves, et al., Controlling stomor being cool? Plant Sci. (2016), http://dx.doi.org/10.1016/j.plantsci.20

nd the interaction with other stresses [1–3]. In Mediterraneannd some semi-arid sub-tropical climates, terminal drought (thatccurs late in the crop cycle) is common, due to scarce seasonal rain-

Abbreviations: Amax, maximum net photosynthesis; A, net leaf carbon assim-lation; VPD, air vapor pressure deficit; E, transpiration; Enight, Night-timeranspiration; gs, conductance for H2O vapor andCO2; gsmax, maximum leaf diffusiveonductance; gs, conductance for H2O vapor and CO2; gsmax, maximum leaf diffusiveonductance; gsnight, Night-time conductance for H2O vapor; RV, resident vegeta-ion; S, pore size; SD, stomatal density; ST, soil tillage; WUE, water use efficiency;

UEi, intrinsic water use efficiency; WUEl, instantaneous water use efficiency;UEc, season-long crop water use efficiency; ∂E/∂A, marginal unit water cost of

lant carbon gain.∗ Corresponding author.

E-mail address: mchaves@itqb.unl.pt (M.M. Chaves).

ttp://dx.doi.org/10.1016/j.plantsci.2016.06.015168-9452/© 2016 Published by Elsevier Ireland Ltd.

fall or to limited pre-seasonal stored soil moisture. Often, terminaldrought co-occurs with high air temperature and high air vaporpressure deficit (VPD) [4]. The great variability of the weather thatcharacterizes semi-arid climates amplifies the potential of stressthat plants may be subjected to. The situation is likely to be exac-erbated by an enhanced frequency of extreme events induced byclimate change, as is the case of heat waves [5].

Most crops cultivated today have been selected for optimalperformance under the current climatic conditions and have pro-gressed towards the yield potential with the green revolution[6]. Yield potential determines crop production in the absenceof drought. However, with the increased risk of water shortage,sustainable production systems are being developed (selection ofgenotypes and agronomic management tools), where irrigationwater and nutrients are used with parsimony [7]. Under thesecircumstances, the basic drought mechanisms – drought escape,avoidance or tolerance – become vital for crop production. Drought

atal aperture in semi-arid regions—The dilemma of saving water16.06.015

escape through changing phenology or sowing/planting date isdesirable in the case of predictable drought. Avoidance and toler-ance traits are important under both predictable and unpredictabledrought scenarios.

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Matching crop water demand with season supply of water willnable crops to escape terminal water stress [3]. Increased cropater use efficiency (WUE), defined as the ratio of leaf carbon

ssimilation (A) to transpiration (E), may be important to saveater for the crucial periods of plant development [8], but has to be

quated in terms of the balance between water savings and yieldenalty [2]. Moreover, when drought and heat co-occur stomatallosure and decreased transpiration, associated with high WUE,ay lead to a dramatic increase in leaf temperature (up to 7 ◦C

bove air temperature) [4]. If this situation stands for long periodseaf photo-damage and/or xylem embolism may occur, leading toevere defoliation and plant death.

Stomata play a central role in the pathways for both carbonptake and water loss by plants. Regulation of stomatal aperture,stimated by stomatal conductance, is a complex process withperational limits set during leaf development, namely the patternf stomata in epidermis that include distribution, size and densitynumber per unit area of leaf) [9]. Recent advances in molecu-ar genetics of stomatal development together with the physicaliffusion model of stomatal conductance revealed a direct rela-ion between the physiology of stomata and the role of genesegulating stomatal pattern [10]. This regulation is dependent oneveral environmental stimuli [11], signaled via internal factorsuch as hormones and hydraulics [12–15]. Under high atmosphericvaporative demand, stomatal response to VPD is an importantechanism to save water, showing genotypic differences, even

etween closely related species [16] that can be explored by plantreeders. Similarly, the sensitivity of stomata to dehydrating soil

s variable, explaining the isohydric or ‘pessimistic’ response asompared to the anisohydric or ‘optimistic’ (but riskier) response.sohydric species are likely more susceptible to xylem cavitationnd therefore tend to have stricter control of transpiration, whereasnisohydric tend to use available water in a less conservative waynd have presumably a lower risk of xylem embolism [17].

In this paper we discuss how stomata are regulated under aariable environment, how the type of stomatal responses influ-nces crop WUE and leaf/canopy temperature and ultimately howlant breeding and management can improve crop performancender hot and dry conditions to resolve the dilemma between sav-

ng water or being cool. We use examples from our own work inrapevine, a species with high genotypic diversity, including vari-ties exhibiting iso- and aniso-hydric stomatal control and whichs cultivated under rainfed and irrigated conditions. This perennialrop is a recognized model to study plant water relations [18].

. Stomatal regulation of water loss

.1. The carbon compromise

Stomatal regulation of carbon uptake and water loss under ahanging environment was a key step in the colonization of landy plants [19]. The evolutionary pathway of this regulation is still

argely unknown [20], but may have been an incremental one, overore than 450 million years since stomata first evolved [21]. It

mplied a ‘carbon compromise’: to fix CO2 from the atmospherehile avoiding lethal dehydration of the photosynthetic leaf tis-

ues. An impermeable cuticle on leaf epidermis would preventater vapor loss to the atmosphere, but would not allow CO2

ptake as well. Stomata are the microscopic pores that provide aariable porosity in the epidermis. They were described for the firstime over three centuries ago and begun to be studied with mod-

Please cite this article in press as: M.M. Chaves, et al., Controlling stomor being cool? Plant Sci. (2016), http://dx.doi.org/10.1016/j.plantsci.20

rn methods in the last years of the 19th century. Francis DarwinCharles Darwin’s son) for example, recognized that stomata closedn response to plant water deficits [22]. Since then, there was vastrogress in our understanding of stomatal functioning, from the

PRESSnce xxx (2016) xxx–xxx

mechanics of opening and closing to the behavior of stomata inthe field. In plants with adequate water supply stomata may regu-late leaf temperature close to the optimum for metabolic processes,including photosynthesis [23] or prevent tissue heat damage underexcessive radiation or temperature. A remarkable example of thecooling effect of leaf transpiration was shown by O.L. Lange [24] inthe hot and dry environments of the Mauritania Sahara, where largeleaved species (Citrullus colocynthis) were able to keep leaf tem-perature below the upper temperature limits for survival. Becauselarge leaves have a low convective heat exchange with the air theywould not survive without transpiration cooling. This apparentlyparadox phenomenon (existence of large-leaf species in deserts,instead of small-leaved ones that intercept less radiation) was alsoobserved by Smith [25] in the Sonoran desert of southern Califor-nia. He recorded leaf temperatures of large leaved desert perennials20 ◦C below air temperature (40 ◦C or above). This was explainedby very high rates of transpiration made possible by plants takingadvantage of sporadic rainfall events.

Stomatal aperture, measured as the conductance (gs) for CO2 orwater vapor, varies continuously with changes in the environment(light intensity, atmospheric CO2 concentration, air temperature,air humidity, wind) as well as with time of day and plant water sta-tus [11]. Therefore, the term of reference will be the maximum leafdiffusive conductance (gsmax), which depends on pore size (S) andthe number of stomatal pores per unit leaf area (stomatal density,SD). Other characteristics (mainly anatomical) such as the positionof guard cells relative to the epidermal cells (e.g. sunken stomataas in Pinus and other conifers, or Nerium or deposition of waxeson the stomata) may reduce gsmax. There is a set of conditions thatinduce changes in stomatal size and density of stomata. For exam-ple, the increase in CO2 concentration in the atmosphere stimulatesthe production of leaves with less number of stomata per unit leafarea and vice-versa in lower CO2 environment [26]. The impact,however, may not be large as there is experimental evidence thatdensity is negatively correlated with stomata size [27].

2.2. Water use efficiency

Plants differ in the amount of carbon assimilated per unit of massof water lost, i.e. their instantaneous water use efficiency (WUEl)that can be estimated as the ratio of leaf net carbon assimilation(A) to transpiration (E), i.e. A/E (in mmol CO2 mol−1 H2O). The ratioA/E is highly variable with environmental conditions, namely vaporpressure deficit (VPD) that determines the transpiration rate. Thatmeans that comparison of plants under different climatic condi-tions cannot be done. In order to avoid ambiguity associated withthe effects of VPD, we may use instead the ratio of carbon assim-ilation to stomatal conductance A/gs referred to as intrinsic wateruse efficiency (WUEi) [28,29]. However, often we want to know theWUE encompassing a growing season of a crop. A season-long cropwater use efficiency (WUEc, g DM kg−1 H2O) can then be defined asthe ratio of the net gain of plant biomass (dry matter) over a givenperiod, by the water lost over the same time.

When comparing C3 and C4 species it is apparent that C4plants exhibit higher WUE due to higher Amax and lower gsmax.In warm regions, where C4 species evolved, photorespiration wasstimulated considerably, as well as transpiration demand [30]. Byincreasing CO2 concentration nearby RuBisCO C4 plants greatlyenhanced carboxylation efficiency and were able to inhibit pho-torespiration. By producing smaller stomata (for a given stomataldensity) or reducing stomatal aperture plants will function atlow gsmax. Lower gs will improve plant water status and mitigate

atal aperture in semi-arid regions—The dilemma of saving water16.06.015

hydraulic demands on the conducting pathway in the xylem, there-fore preventing hydraulic failure [31].

In general, stomatal regulation of gas exchange at the leaf leveloperates in a way that maximizes carbon assimilated per water

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ranspired. Optimum WUE involves stomatal restriction of leafranspiration to the periods when evaporative demand is high-st. That is why stomata are more closed at midday and in thefternoon than in the morning [11]. In a daily basis, the lower theater available (or the higher the value of VPD), the lower the time

llocated to carbon assimilation (stomata open). In stressed plantshotosynthesis may be limited to the morning hours, before VPD

s too high [23,32]. Another example of optimization in leaf A/E ishe effect of wind. Increasing wind speed enhances carbon diox-de (CO2) assimilation while reducing transpiration due to morefficient convective cooling (under high solar radiation loads), thusmproving WUE [33].

In the attempt to reach a consensus theoretical model for sto-atal conductance regulation an economic analogy was proposed

34] based on the hypothesis that gs is regulated to minimize themount of water spent to fix one unit of carbon, i.e. an optimalityroblem. In economics the marginal cost represents the total cost

or producing one more unit. In this case, the marginal unit waterost of plant carbon gain (∂E/∂A) is constant over a specified timerame. In other words, the ratio of the sensitivities of E and A tohanges in gs [i.e. (∂E/∂g)/(∂A/∂g)] remain constant over the spec-fied time frame [34], or to put it in a more colloquial statement,arbon gain is maximized for a given amount of water loss [35].

.3. Night-time transpiration – significance and impact on WUE

Even though the optimization models seem to apply to manyituations [35] there are apparent inefficiencies. One example ishe existence of night transpiration, due to incomplete stomatallosure during the night, which involves the loss of water in theark without carbon assimilation. Night-time transpiration (Enight)an result in a significant water loss and in a major reduction of

UE, as reported for both crop species [36–38] and model plants39–41]. Rates of Enight are documented across a wide phylogeneticnd ecological number of species, (e.g. genus Rosa) and are gener-lly low, within the range of 5–15% of day transpiration [36,38,42]ut they may reach higher values (up to 25% to 30%) in dry environ-ents [36,43]. Konarska et al. [44] found that Enight was observed

n several forest species, and amounted to 7 and 20% of midday of sunlit and shaded leaves, respectively. Other studies describeariation on gsnight between different genotypes [45–47] and alsoetween closely related species [40,48]. Also important is the facthat Enight may reduce WUE at the landscape level and be relevantor the closing of water balance, locally and globally [49]. In gen-ral, after stomatal closure in the beginning of the night, leaf gasonductance start to increase but the maximum of Enight occurs inhe hours just before dawn [50]. The time sequence of nocturnaltomatal closure suggests an endogenous regulation, possibly asart of circadian rhythms rather than a direct response to changes

n VPD or in temperature. Night-time stomatal opening may bedaptive for the plant with the circadian clock providing an antici-ation of sunrise, when carbon assimilation will start. High valuesf predawn stomatal conductance mean that leaves are ready toake full advantage of the first morning sun light to fix carbon whilePD and temperature are still low. Later in the day, conditions are

ess favorable and WUE is reduced. This is of great importance inemi-arid environments.

The mechanisms regulating Enight in plants remain unclear [49].t is known that gsnight responds to similar internal and exter-al factors as daytime transpiration, e.g. ABA, CO2 concentration,ind speed, drought stress and VPD [36,37,51]. Also, in Arabidopsis

haliana mutants with abnormal stomatal closure in darkness (open

Please cite this article in press as: M.M. Chaves, et al., Controlling stomor being cool? Plant Sci. (2016), http://dx.doi.org/10.1016/j.plantsci.20

ll night long – opal) stomata close normally in response to ABA andtmospheric CO2 [41]. This suggests that the response of stomatao light to dark transition can be partly decoupled from ABA or CO2ignaling pathways. It is possible that selection for high gsnight and

PRESSnce xxx (2016) xxx–xxx 3

related water loss may have occurred in habitats characterized byabundant water resources but low nutrient availability. This mayindicate that Enight would be more related to nutrition than to a“cooling function” in plants.

2.4. Regulation of stomata movements at the molecular level

Regulation of stomatal movement is one of the most studiedmodel systems for cellular signaling transduction and involvesmany proteins responsible for controlling stomatal responses tothe environment [52,53]. This regulation is exerted either via stom-ata density and pattern or via stomatal aperture. Among the genesknown to affect stomata functioning is ERECTA gene family. ERECTAare putative leucine-rich repeat receptor-like kinases that havebeen related to the perception of drought stress signals across cellmembrane in the mutant of Arabidopsis with improved transpira-tion efficiency [54]. ERECTA are known to affect stomata patterning[55], which will have implications on CO2 diffusion through themesophyll, with impact on photosynthesis [54]. ERECTA genes areconsidered to improve WUE [56] [45] and increase drought tol-erance [57]. But they also exert pleiotropic effects, independentlyfrom the effects on plant water status, which includes a role in thecircadian clock and in thermotolerance improvement [55,58].

On the other hand, several proteins located in the plasma mem-brane and tonoplast of guard cells, including channels and carriersare known to be involved in the control of stomata movements[41,53,59–61]. The activity of these proteins is regulated via post-translational modifications, particularly via phosphorylation [62].Several of these proteins play signal transduction roles duringplant adaptation to stress, with an involvement ranging from stresssignal perception to stress-responsive gene expression [63]. Theexpression of these responsive genes is known to be regulatedby ABA-dependent and ABA-independent pathways [64]. Althoughseveral studies report the convergence between both pathways[65] the knowledge on how the two signaling pathways regulateeach other is limited [63].

ABA induced stomata closure is related with protein modifica-tion via PP2C (type 2C protein phosphatases), which affects theactivity of several channels and carriers. The ABA perception bythe guard cells is undertaken by several ABA receptors involvedin the network of ABA responses, namely RCAR/PYR1/PYL-PP2Ccomplexes. When the complex binds ABA [58], inhibition of thePP2C activity occurs and targets are phosphorylated. ABA signalingtransduction regulating stomatal response involves several genes[66] and evidence showed that plant membrane transport systemsplay a significant role in this response, either passively throughchannels and carriers or actively by primary and secondary trans-porters using ATP hydrolysis and ions gradients to drive solutesacross membranes [67]. ABA is imported into guard cells by ABCG40transporter, activating the S-type (slow-activating sustained) anionchannels and the R-type (rapid-transient) anion channels, whichfacilitates the efflux of anions such as malate2−, Cl−, and NO3− seereview [60].

An elevation of cytoplasmic Ca2+ concentration due to Ca2+-permeable channels located in the tonoplast and the plasmamembrane is also observed during ABA dependent stomatal clo-sure [68,69]. These proteins are encoded by genes belonging tothe TPC1 (two-pore channel 1), CNGC (cyclic nucleotide-gatedchannel), and GLR (glutamate receptor-like) families [66]. Simul-taneously, H+-ATPase and inward K+ channels (KAT1/KAT2) areinhibited, inducing a depolarization of the plasma membrane [70]and the activation of the efflux of potassium, which is facilitated

atal aperture in semi-arid regions—The dilemma of saving water16.06.015

by GORK (guard cell outward rectifying K+) channels [71–73].The inhibition of GORK activity in guard cells results in defectsin K+ efflux and lower stomatal closure [72]. More recently, astress-responsive K+ uptake permeases (KUPs), sharing redundant

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unctions with GORK channels were identified [74]. KUP6 protein,n A. thaliana K+/H+ symporter, localized to the plasma mem-rane of guard cells, is highly up-regulated in response to the ABAreatment and water deficit. However, Becker and colleagues [71]bserved an up-regulation of GORK transcription upon onset ofrought in guard cells to be ABA insensitive. These data shows thatlants have tools to adjust stomatal movement in an ABA inde-endent way. ABA independent pathways have been elusive buthere are evidences supporting the role of another hormone (sali-ylic acid) and gas molecules as NO, CO and H2S in these pathways75,76].

Aquaporins are also considered to have a role on the regula-ion of the opening and closure of stomata, namely by providinglants with the means to rapidly and reversibly modify water per-eability [77,78]. A recent report [79] shows the direct role of a

pecific Plasma membrane Intrinsic Protein (PIP2;1) in stomatalovement. It is suggested that the ABA-triggered stomatal closure

equires an increase in guard cell permeability to water, throughST1-dependent phosphorylation of PIP2;1.

In addition to the regulation of protein activity, evidencehows that transcriptional factors are also involved in membraneransport in guard cell signaling, therefore controlling stomatal

ovement. At guard cells, several TFs regulate stomatal openingn an ABA dependent way [80]. Different R2R3MYB family mem-ers controlling either dark-light or ABA pathways and involved

n the opening or the closure of stomata were identified. Particu-arly AtMyb60 is involved in stomata opening in the light, as wells in the ABA and desiccation signaling perception. A functionalrtholog to AtMyb60, from grapevine VvMyb60, was shown to beBA sensitive [81]. Several other transcription factors families were

dentified acting as repressor or positive regulators of drought resis-ance [53,80].

The last decades provided the community with a large body ofata not only of highly scientific relevance but also with breed-

ng potential [61]. There are still some gaps to be closed but theenes that code for the proteins involved in stomata movementnd regulation are excellent candidates for breeding programs.

. Leaf and canopy temperature

.1. Regulation of leaf temperature

Leaves only absorb a small amount of the incident radiation androm this absorbed energy most is dissipated as sensible or as latenteat (transpiration). In well-watered plants, transpiration repre-ents the most effective way of leaf cooling. Under water deficits,ith stomata closed, transpiration only occurs via cuticle. Further-ore, cuticular transpiration increases exponentially with rising

emperature due to the increase in the water permeability of cuticlend in VPD [17].

Regulation of leaf temperature is strongly dependent on leaforphology, i.e., leaf size and shape [82]. These traits are highly

ariable and likely to be influenced by different selection pressureactors. They are determinant in plant’s growth performance inerms of the response to heat, water and light stress [11,82–84].lants in drier climates tend to present smaller leaves, whereas

arger leaves are more common in humid climates [84]. Indeed,nergy balance models predict that leaf temperature is higher inarge canopy leaves due to thicker boundary layers for a given radi-tion and wind speed.

The relevance of stomata in leaf cooling is especially recognized

Please cite this article in press as: M.M. Chaves, et al., Controlling stomor being cool? Plant Sci. (2016), http://dx.doi.org/10.1016/j.plantsci.20

n tall canopies like fruit and forest trees. However, even in dwarfanopies the higher stomatal density and gsmax than it would beredicted on the basis of regulating transpirational water loss sug-ests the need for transpirational cooling [85].

PRESSnce xxx (2016) xxx–xxx

As a result of a combination of large influx of absorbable energywith insufficient loss of heat, leaf overheating may take place, lead-ing to a down regulation of photosynthesis or photoinhibition. Ingeneral, high temperatures only last a few hours of the day, so inmost cases plants can overcome it without major negative effects.However, if the condition prolongs and ‘lethal limits’ are crossed,permanent injuries such as leaf necroses may occur. Thresholds forheat damage are characteristic of the species but vary with organsand tissues. These threshold temperatures can vary from 45 to 50 ◦Cin temperate zone species to 50–65 ◦C in sub-tropical C4 grasses andwoody plants as well as in grasses of the steppes [83]. Increasedtemperature is known to accelerate leaf aging and senescence asa result of high ABA and reduced cytokinin content produced inwilting leaves [17].

3.2. Optimal temperature for leaf functioning. Thresholds for leafsurvival

The interval of leaf temperatures for attaining maximum photo-synthesis vary with species, ranging from 20 to 35 ◦C in temperatezone C3 crops to over 35 ◦C for C4 species, whereas the optimum forgrowth is somewhat lower [11]. The higher optimal temperaturefor carbon assimilation in C4 plants results from the inhibition ofphotorespiration induced by the CO2 concentrating mechanisms inthe bundle-sheaths they acquired during evolution.

Optimal temperatures for carbon assimilation are also depen-dent on the environmental conditions during the growth periodthat may induce plant’s acclimation to the particular weather con-ditions of the season [23,53].

Biochemically, high temperatures pose several challenges tocells, namely to the photosynthetic-related enzymes. Cells have toensure organelle stability, deal with unfavorable O2 to CO2 ratiosand unfavorable oxidative status that decrease both the rate of CO2fixation and the activity of the enzymes of the Calvin cycle. Photo-synthesis occurs in different sub-compartments of the chloroplast,which are metabolically distinct. Organelle stability (membraneintegrity and fluidity) is essential in the control of the biochemi-cal environment and therefore protein activity. High temperatureis typically associated with a higher potential for O2 production(via high light). Furthermore, the relative solubility of O2 to CO2increases with temperature, leading to a higher competition forRuBisCO catalytic site and lower CO2 fixation. Higher O2 concentra-tion also has the potential to disturb the oxi-reduction homeostasisin the cell, further contributing to lower carbon assimilation sinceseveral enzymes of the Calvin cycle are only active when reduced[59].

Temperature also influences the rate of enzymatic reactions,exerting a positive effect until a denaturation threshold is achieved,which causes enzyme inactivation. Super-optimal temperatures(above such threshold) lead to enzyme inactivation. For mostenzymes, the temperature denaturation threshold is unknown.Koning [86] describes that most enzymes are denatured above50 ◦C and that the optimum temperature for typical enzymes iscomprised between 40 and 50 ◦C. As an exercise, the optimum tem-perature of several photosynthetic-related enzymes was searchedin the enzyme database BRENDA (version 2015.2) [87]. We alsosearched for ferredoxin and ferredoxin-thioredoxin reductase, keyplayers in keeping Calvin cycle enzymes reduced and thereforeactive. Considering those enzymes with more than four repre-sentatives (Table 1), it was found that the range of optimumtemperatures is wide. The 35–40 ◦C range is a cornerstone for pro-tein activity and denaturation, with strong impact on the carbon

atal aperture in semi-arid regions—The dilemma of saving water16.06.015

assimilation and redistribution within the plant, in both C3 and C4species.

The optimum temperature is calculated using purified enzymesand may not reflect the in vivo inactivation threshold. For exam-

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Table 1Range of optimum temperatures and median optimum temperatures for some photosynthetic-related enzymes in higher plants available at the BRENDA(BRaunschweig ENzyme Database), database, using the tool “functional enzyme parameters” (www.brendaenzymes.org/statistics.php?valueSelect=TemperatureOptimum&taxTerm=&ecNumber=). We searched for several enzymes (see table footnote) but for this table we considered only the enzymes with more than four entries atthe database: RuBisCO – C3 & C4; Phosphoglycerate kinase – C3 & C4; PEP carboxylase – C4 & CAM; Ferredoxin – regulatory protein which activates several photosyntheticenzymes via protein reduction. The activity of Ferrodoxin depends on the photochemical reactions.

Enzyme EC number N◦ entries in BRENDA Range of optimum temperatures Median optimum temperatures

RuBisCO 4.1.1.39 11 25–40 ◦C 25 ◦CPhosphoglycerate kinase 2.7.2.3 5 22–30 ◦C 25 ◦CPEP carboxylase 4.1.1.31 15 25–67 ◦C 30 ◦CFerredoxin 1.8.7.1 4 22–37 ◦C 33.5 ◦C

Footnote: Enzymes considered: Calvin cycle: phosphoribulokinase [EC:2.7.1.19], ribulose-bisphosphate carboxylase-oxygenase [EC:4.1.1.39], phosphoglycerate kinase[EC:2.7.2.3], glyceraldehyde-3-phosphate dehydrogenase [EC:1.2.1.13], fructose-bisphostolase [EC:2.2.1.1], sedoheptulose-bisphosphatase [EC:3.1.3.37], ribose 5-phosphate isomcarbonic anhydrase [EC:4.2.1.1]; cellular redox homeostasis dependent of the photochemic

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Fig. 1. Light response curves for net photosynthesis (A) and stomatal conductanceto water vapor (gs) measured for three Vitis vinifera L varieties: ‘Touriga Nacional’,‘Syrah’ and ‘Cabernet Sauvignon’ subjected to deficit irrigation in the field, in Alen-tejo. Vines were 6–7 years old and were grafted on the rootstock 1103-Paulsen. Gasexchange measurements were done with a portable infrared gas analyzer (Li-cor6400, Li-Cor Inc.) with a light chamber 6400–02 B (Li-Cor Inc.) equipped with a LEDred/blue light source. The light curves were obtained by measuring A and gs at steadystate under different PPFDs (2000–0 �mol m−2 s−1), at constant air CO2 (360 mL L−1),a T block set at 25 ◦C and an air flow rate set at 500 mmol s−1. Measurements weredone between 9.00 and 13.00 h in August 2006, on 4–6 plants per variety, one leafper plant. Under the moderate water stress conditions of the trial (�pd around –0.5 MPa), ‘Touriga Nacional’ had higher values of stomatal conductance to watervapor and lower Tleaf than the ‘Syrah’ and ‘Carbernet Sauvignon’, with 33.9 ± 1.0 ◦CfLf

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or ‘Touriga Nacional’, 36.9 ± 0.2 ◦C for ‘C. Sauvignon’ and 35.6 ± 0.2 ◦C for ‘Syrah’.eaf gas exchange and temperature values are means ± SE (n = 4–6 plants). Adaptedrom Costa and co-authors [129].

Please cite this article in press as: M.M. Chaves, et al., Controlling stomor being cool? Plant Sci. (2016), http://dx.doi.org/10.1016/j.plantsci.20

le, it was shown that the RuBisCO deactivation state is caused byeduced activity of RuBisCO activase, which is more sensitive toemperature than RuBisCO itself [88]. In the last decades a largeody of knowledge has been generated illustrating the several lev-

phate aldolase [EC:4.1.2.13], fructose-1,6- bisphosphatase [EC:3.1.3.11], transke-erase [EC:5.3.1.6]; C4 enzymes: phosphoenolpyruvate carboxylase [EC: 4.1.1.31],al reactions: ferredoxin [EC:1.8.7.1]; ferredoxin-thioredoxin reductase [EC:1.8.7.2].

els of protein activity regulation [59]. The events taking place atprotein level will determine protein activity and pathway fitnessas a single enzyme can block the full pathway (i.e. the protein withlowest temperature threshold). Such information, when combinedwith kinetic studies, is highly relevant to the understanding of cropproductivity under challenging temperature. However, the wholeregulatory network needs to be deciphered, in order to identifytemperature QTLs and their potential impact. Such information canbe used in breeding strategies for more efficient photosynthesisunder temperature constraints.

4. Genotypes – a role for breeding?

Given the expected water restrictions, in particular for Mediter-ranean environments and some semiarid subtropical climates,genotypes that are able to compromise between high WUE andleaf cooling capacity are required. To avoid thermal damage, thebest adapted genotypes must have the ability to maintain stom-ata open and transpire when optimum temperature is exceeded,therefore benefiting from leaf evaporative cooling [89]. Evaporativecooling can be promoted under irrigation. In rainfed crops a strictcontrol of stomatal aperture to prevent xylem cavitation may leadto supra-optimal leaf temperatures, therefore requiring resilientmetabolism towards heat stress.

There is evidence for genetic variability in traits that controlstomatal response to VPD, with impact on performance under heatand drought conditions. For example, in some soybean genotypesthe response of leaf transpiration to VPD was found to be linear atlow VPD but slowing down until a plateau was reached for a givenVPD threshold. This allows soil water conservation during periodsof low rainfall [8]. Synergism between soil water deficits and VPDon reducing stomatal aperture was also reported, for example, intrees of the Mediterranean (Quercus ilex and Quercus suber) in whichstomatal response to VPD was more pronounced during summerdrought than under pre-drought conditions [16].

The temperature regime during canopy development can alsoinfluence the upper limit of canopy conductance via stomatadensity and size. Long-term effects of a higher thermal regimeduring the growing season were studied in grapevines, showingan enhanced gs as a result of longer and wider open stomata, ascompared to plants grown at a lower temperature regime [90].

4.1. Iso- and aniso-hydric responses

Differences in stomatal sensitivity to water deficits betweenspecies or cultivars may serve to compensate for differences in the

atal aperture in semi-arid regions—The dilemma of saving water16.06.015

vulnerability of xylem to cavitation [91]. According to the type ofstomatal regulation in place, plants have been classified as pre-senting an isohydric or ‘pessimistic’ stomatal behavior, in contrastwith the anisohydric or ‘optimistic’ behavior, where a more fluc-

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Fig. 2. View of a grapevine canopy where two red grape varieties (‘Touriga Nacional’and ‘Cabernet Sauvignon’), grafted on the same rootstock (SO4) are grown side byside in a non-irrigated vineyard located at the Lisbon winegrowing region (TorresVedras), in Portugal. Image taken at the ripening period after a dry season withhNt

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eat waves, showing a higher senescence and/or leaf burn on the variety ‘Tourigaacional’ (characterized by a higher stomatal conductance to water vapor relatively

o ‘Cabernet Sauvignon’).

uating water potential in response to dehydration is allowed [92].his classification does not reflect a strict genotypic character sincehe growing conditions (e.g. field versus greenhouse, potted vsoil grown) and the degree of imposed stress also modulate theesponse [93].

The anisohydric response involves the use of soil wateresources by the plant until lower water potentials are attainedhan in the isohydric case. This is due to higher stomatal con-uctance/leaf transpiration, with plants of the anisohydric groupresenting cooler leaves and higher photosynthetic rates than iso-ydric ones (Fig. 1). However, a negative side of it can occur – theselants may suffer a rapid dehydration under hot spells due to highranspiration rates not compensated by a fast enough soil waterptake, resulting in the senescence and dry out of basal leaves ofhe canopy (Fig. 2). On the other hand, a more conservative responseis-à-vis the decrease in soil water is exhibited by isohydric plants,ith stomata showing a quick and sensitive reaction to dehydra-

ion. This enables fluctuations in plant water potential in responseo soil water deficit to be minimized and maintained above (lessegative) the critical value for xylem cavitation for longer peri-ds. This protective response will have costs in terms of lower CO2ssimilation rates and reduced growth [93] and under heat-waveituations leaf temperatures are likely to attain critical thresholdsor leaf damage.

The different response to water scarcity is also associated withifferent root/shoot ratios, with anisohydric stomatal behavioreing often associated with larger root systems and high capacityor osmoregulation that support water uptake until low soil waterontent [4]. Osmoregulation requires a dehydration signal to beeveloped, more likely to take place in aniso- than in isohydriclants. The anisohydric strategy will allow a closer match betweenater availability and consumption, with a positive impact on sea-

on growth.Differences in the signals responsible for stomatal control

Please cite this article in press as: M.M. Chaves, et al., Controlling stomor being cool? Plant Sci. (2016), http://dx.doi.org/10.1016/j.plantsci.20

etween iso and anisohydric plants have been identified [17,92].t was shown that in addition to its direct action in stomata, ABA

ay induce closure of aquaporins in bundle sheath cells, decreas-ng water flow to the mesophyll cells and therefore reinforcing the

PRESSnce xxx (2016) xxx–xxx

effect on stomata with a hydraulic component [91]. This multipleeffect of ABA might explain the more sensitive response to waterdeficits in isohydric plants as compared to anisohydric ones [94,95].This is very important in plants with greater susceptibility to xylemcavitation.

4.2. Roots and rootstocks

Differences in plant responses to water stress have been asso-ciated to differential adjustments in fine root hydraulic physiologyand suberization [96]. Roots which are able to maintain high roothydraulic conductivity under water stress conditions are also ableto maintain greater water supply to the shoot [97]. Indeed, long-distance water transport from roots to leaves is highly dependenton maintenance of xylem functionality.

Recently, it was reported that ABA might exert a control on leafhydraulic conductance, with an indirect effect on stomatal con-ductance [94]. Nonetheless, ABA cannot fully explain the controlof stomatal conductance [98] and recent reports demonstrate therole of aquaporins in root-specific hydraulics [99]. Gambetta andcolleagues [100] observed an up-regulation of several aquapor-ins both under well-watered and drought conditions, associatedwith high root hydraulic conductance. Some aquaporins were alsoreported to have a significant link with stomatal conductance andleaf hydraulic conductivity under mild water deficits [101,102]. Itwas further observed that plant senses the first stages of the stressby increasing root hydraulic conductance, stimulating root-to-shoot chemical signaling, increasing xylem sap pH and modulatingaquaporin expression [102].

Perennial fruit crops are often grafted onto rootstocks that pro-vide better resistance to biotic and abiotic stresses as well asmaintain the desirable qualities of harvested fruits. As in ownroots, rootstock effects on scion are mediated by either chemicalor hydraulic signaling [103,104]. Scion transpiration rate and itsacclimation to water deficit are controlled by the rootstock throughdifferent and complex genetic architecture [104]. Nonetheless, atearly stages of development, the scion appears to be the main driverof shoot growth and biomass allocation in grafted plants [105].Grafting the same variety in different rootstocks significantly alteroverall scion physiological performance, as shown for example ingrapevine [104,106] and in other woody species [107,108].

The adaptation of rootstocks to drought and its influence onthe scion is a complex trait under the control of several phys-iological and molecular processes. Although there are evidencesfor the contribution of rootstock to the genetic variability of WUE[109], differences among rootstocks are not always confirmed infield experiments [110]. Until now, rootstocks are classified qual-itatively. Nonetheless, the single report on the genetic control ofdrought tolerance conferred to scion by the rootstock [104] iden-tify several quantitative loci for scion transpiration and WUE. Thisidentification of rootstock QTL related to improved WUE wouldfacilitate breeding for improved drought tolerant rootstocks. Unfor-tunately, the identification of these genes is being retarded by thelow mapping resolution of such analyses and the number of distinctgenotypes in the population used. Further research is still needed,to improve the understanding of rootstock contribution to waterstress adaptation, in particular in the context of the ongoing climatechange [109].

4.3. Is there an ideotype for semi-arid zones?

Under semi-arid regions an ‘ideal’ crop should be able to

atal aperture in semi-arid regions—The dilemma of saving water16.06.015

combine some of the following strategies: to capture soil waterefficiently, to move more of the available water through the crop(rather than being wasted as soil evaporation or drainage), to exertan efficient stomatal regulation in response to soil or atmospheric

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A 0 5 10 15 20 25 30 35 40

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th (c

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Volumetric soil moisture (%)

0 5 10 15 20 25 30 35 40

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100908070605040302010

April 5thJune 7th

STB 0 5 10 15 20 25 30 35 40

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RV

Volumetric soil moisture (%)

5 15 25 350 10 20 30 40

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th (c

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April 26thJune 6th

ST

Fig. 3. Effect of floor management practices on soil water depletion curves measured in situ during Spring, on two sites, at the third year after experiment setup of the trial.( ated wE l, 200i etatio

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A) Grapevine variety ‘Cabernet Sauvignon’, grown in Alenquer, a coastal non-irrigstremoz, Alentejo, an irrigated hot and dry winegrowing region in South Portuga

nstalled in the row between two contiguous vines. ST – soil tillage; RV – resident veg

ater deficits and/or have an improved crop transpiration effi-iency [4,111].

Irrigation changes substantially crop responses to the environ-ent. Under irrigation, genotypes should benefit from being closer

o the anisohydric type (as defined above), using the available watern order to have open stomata and maintain higher carbon assimi-ation rates until lower soil water content. These genotypes are ineneral resilient to xylem cavitation and need a large and vigor-us root system. In the case of using deficit irrigation strategies,here water supply may be well below total evapotranspiration

f the crop, close monitoring of canopy temperature is desirable,specially in periods when heat waves can occur and crops wereot acclimated to high temperatures.

It is interesting to refer the case of cotton and bread wheat thatere bred for higher yields at supra-optimal temperatures under

rrigation. Successive commercial releases of both crops showedenetic-controlled increases in stomatal conductance that wereccompanied by yield increases [112,113]. It was demonstratedhat stomatal response to temperature (not to light or VPD) was theesponsible factor for separating low and high-yielding cotton lines.hose changes in gs were independent of changes in photosyn-hetic rates and rather associated with leaf cooling, thus providing

valuable avoidance of supra-optimal temperatures [113].On the other hand, isohydric behavior is adapted to rainfed sit-

ations due to highly sensitive stomata to VPD and/or soil watereficits. However, these genotypes will require thermal resiliencet the mesophyll level, to sustain periods of supra-optimal tem-erature without major leaf damage [4]. A conservative patternf water use, rather than deep or profuse rooting as in anisohy-ric crops, is critical for the terminal drought tolerance of theserops. For example, drought tolerant genotypes of chickpea showed

lower water uptake and a lower stomatal conductance at the vege-ative stage than sensitive ones, while tolerant genotypes extracted

Please cite this article in press as: M.M. Chaves, et al., Controlling stomor being cool? Plant Sci. (2016), http://dx.doi.org/10.1016/j.plantsci.20

ore water than sensitive genotypes after flowering [114]. In otherords, higher seed yield corresponded to genotypes that were able

o save water early in the season and use it in late season.

inegrowing region of Portugal, 2004; (B) Grapevine variety ‘Aragonez’, grown in6. Each point is the mean of measurements made on 12 (A) or 16 (B) access tubesn. Adapted from Monteiro and Lopes (2007) [92] (A) and Lopes et al. (2011) [99](B).

5. Management for coping with drought and heat stress

The sustainability of modern irrigated agriculture and increasedcompetition for water resources with other economic sectors isforcing agronomists to use water more efficiently [115,116]. Higherair temperatures and less precipitation, together with increasinglimitations in the available water resources in semi-arid agricul-tural areas, led to the adoption of alternative irrigation strategiessuch as deficit irrigation (irrigation below full crop evapotranspi-ration losses) as means to save water via increased transpirationefficiency [93]. Indeed, it is known that mild to moderate waterdeficits may lead to an increase in WUE due to the maintenanceof carbon assimilation while transpiration is reduced by partialclosure of stomata [93].

Other strategies to improve water productivity under irriga-tion involve optimization of the timing and duration of irrigationevents to reduce evaporation and percolation, together with theselection of the most suitable crops/genotypes for specific climateconditions, optimal sowing and harvesting times or precise irri-gation to prevent water deficits, taking into account the weatherconditions and crop growth stage [116,117]. Selection of the mostadequate crops and daily and seasonal irrigation strategies stronglydepend on the eco-physiological and phenological characteristicsof the species and also of the variety. In the case of deficit irrigation,water can be reduced or withhold at specific phenological stagesas it happens with regulated deficit irrigation (RDI). RDI createswater deficits during specific periods of the season to save waterwhile minimizing or eliminating negative impacts on crop revenue[118]. Imposition of a mild water stress to crops enables a bettercontrol of canopy development, avoiding dense canopies, thereforereducing water consumption and improving WUE [119]. However,in warm regions implementation of deficit irrigation must be donejudiciously. When exposed to heat waves, mildly stressed vines

atal aperture in semi-arid regions—The dilemma of saving water16.06.015

become more vulnerable to leaf burn (Fig. 2). Under such condi-tions deficit irrigation should be replaced by full irrigation in orderto increase evaporative cooling [120].

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Together with irrigation, precise plant monitoring is an abso-ute need to cope with the increase in frequency of extreme eventsdrought and heat waves) [116]. Imaging techniques, such as ther-

ography, provides a remote, non-intrusive approach to monitorrop performance and water status at different scales of timend space. Thermography has permitted to overcome limitationsosed by temperature point measurements and to complementhe assessment of soil and plant water status. It is being appliedn modern precision agriculture [121].

Cover cropping is a cultural practice widely used with peren-ial crops in many areas of the world, being recommended toromote environmental sustainability [116,122]. The benefits ofover crops are many, ranging from environmental protection (e.g.ontrol of soil erosion, enhancement of soil structure and biodiver-ity, sequestering carbon) to crop management, including controlf crop vigor and improved fruit composition, as described forxample in grapevine [122]. Despite those potential benefits, thedoption of cover crops in Mediterranean non-irrigated fruit cropsas been limited by the concern of excessive water competitionetween the swards and the crop [123,124]. However, in Mediter-anean and semi-arid regions water competition by the swards isffective only during spring [119,125,126], when favorable tem-eratures combined with high soil water availability can induceigh vegetative growth rates and transpiration of the sward species.uring the summer, sward vegetation dries out, becoming deadulch that will reduce soil evaporation [123]. Interestingly, the

evelopment of deeper roots in the case of vines was observedfter several years of competition with swards, therefore increasinghe capacity for water extraction by the roots in deeper soil lay-rs and the potential for sustained transpiration under heat stress127,128]. Indeed, a higher water extraction from deep soil layersas observed in the treatment with permanent resident vegetation

RV) as compared with the soil tillage treatment (ST) where muchower or almost no water was extracted from deep layers (between.80 and 1.0 m deep), (Fig. 3). These results can be explained by the

ikely lower soil evaporation caused by the mulching effect of coverropping residues during summer [123], by the smaller total cropeaf area in the RV treatment due to competition with swards andhe increased water uptake by crop roots from deeper soil layers, as

result of a compensatory growth of the root system. Summariz-ng, the use of cover crops is a management practice that can have

positive influence on crops water use by preventing excessiveigor, in case water is fully available in spring, or by maximizinghe volume of soil explored by roots through the enhancement ofhe exploitation of soil water reserves into deeper layers.

. Concluding remarks and future perspectives

Basic science has already generated great advances in our under-tanding of plant stress tolerance. The increase in drought and highemperature predicted in climate change scenarios for the Mediter-anean and semi-arid zones still poses great challenges to cropreeding and management in the near future. To deal with thesecenarios information is a most wanted resource, being necessaryo gather and integrate information at many levels.

The inextricable nature of ‘carbon compromise and leaf tem-erature’ dilemma involves a series of interactions between plantsnd the environment and has to be framed into the particulargricultural system being pursued (rainfed or irrigated). In gen-ral, genotypes desirable for semi-arid areas should be able toi) maximize the extraction of available soil water (e.g. vigorous

Please cite this article in press as: M.M. Chaves, et al., Controlling stomor being cool? Plant Sci. (2016), http://dx.doi.org/10.1016/j.plantsci.20

oots/rootstocks); (ii) minimize water loss per unit of fixed carbony stomatal regulation (high WUE) and optimize its use accordingo the phase of the growing cycle; (iii) adjust canopy size to avail-ble water; (iv) show heat-avoidance traits (as e.g. paraheliotropic

PRESSnce xxx (2016) xxx–xxx

movements) or heat-resistance at the leaf mesophyll level and/or(v) maintain cool canopies when irrigated, in order to cope withperiods of enhanced temperatures.

The operational combination of different traits will vary accord-ing to the drought/heat scenarios that we wish to target. Cropmanagement tools are required to complement the use of well-adapted genotypes, such as irrigation and floor managementstrategies, accompanied by close plant monitoring during the sus-ceptible phases of crop development.

Areas of research that should be reinforced include the studyof species thresholds for high temperature and for xylem cavita-tion to guide us in preventing irreversible damages. These typesof studies when performed under controlled conditions should befollowed by field experiments to enable the full assessment of thegenotypes resilience and acclimation mechanisms. Indeed, leavesmay reach temperatures higher than the threshold defined for adecrease in maximum photochemical efficiency and still no pho-todamage be observed [8]. The combination of the intensity andduration of stress will define the final conditions that dictate plantsurvival or failure.

Future research should also provide a molecular basis for animproved understanding of the different strategies that plantsuse to regulate their water status and temperature. Molecularmarkers identified for their association with physiological traits(or their proxies), such as water uptake (roots), water use effi-ciency (stomata and photosynthesis), water status (aquaporins andosmoregulation) or canopy temperature, as important drivers ofplant development under stress conditions, can play a major rolein supporting crop breeding for drought-prone environments.

Acknowledgements

Parts of research presented in this paper have received fund-ing from European Community’s Seventh Framework Programme(FP7/2007-2013) under the grant agreement no. FP7-311775,Project INNOVINE. O. Zarrouk, had a scholarship from INNOVINEand FCT (SFRH/BPD/111693/2015) and J.M. Costa had a scholarshipfrom INNOVINE and FCT (SFRH/BPD/93334/2013).

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